木醋桿菌C5紫外誘變育種及其培養(yǎng)基優(yōu)化.pdf

1、哈爾濱工業(yè)大學(xué)理學(xué)碩士學(xué)位論文摘要細(xì)菌纖維素(BacterialCellulose，縮寫作BC)作為一種新型的生物材料，已經(jīng)在造紙、紡織、醫(yī)藥以及食品工業(yè)中取得成功應(yīng)用。但是工業(yè)生產(chǎn)中由于細(xì)菌纖維素的高成本限制了它的大規(guī)模應(yīng)用。因此我們有必要選育出高產(chǎn)的纖維素產(chǎn)生菌，并對其培養(yǎng)基成分及培養(yǎng)條件進(jìn)行優(yōu)化，以提高產(chǎn)量，降低成本。以木醋桿菌(Acetobacterxylinum)C5為出發(fā)菌株，應(yīng)用L9(34)正交試驗(yàn)設(shè)計(jì)優(yōu)化菌懸液制備的最佳

2、條件。根據(jù)極差分析確定:將木醋桿菌C5在30℃下靜止培養(yǎng)9天，將其形成的纖維素膜在無菌的條件下轉(zhuǎn)入12cm直徑的大平板中，然后加入1.5m1纖維素酶在30℃下酶解12h，制得菌數(shù)達(dá)到2.2x10“個(gè)ml的菌懸液。對木醋桿菌C5進(jìn)行紫外誘變，繪制木醋桿菌C5的致死率曲線，將照射時(shí)間3min作為紫外誘變劑量。從誘變固體培養(yǎng)基上挑起230個(gè)菌落，將這些菌株的產(chǎn)膜厚度與木醋桿菌C5進(jìn)行對比，篩選到72株正向突變菌株。擴(kuò)大培養(yǎng)，復(fù)篩得到產(chǎn)量顯著高

3、于木醋桿菌C5的244468和98號菌株，增幅分別為29.88%46.06%19.50%38.17%。選擇增幅最大的44號突變菌株進(jìn)行下一步實(shí)驗(yàn)，將其命名為木醋桿菌突變株C544o通過單因素試驗(yàn)確定木醋桿菌突變株C544產(chǎn)纖維素的適宜溫度范圍在25310C30℃時(shí)纖維素產(chǎn)量最高，溫度超過33℃時(shí)不再產(chǎn)生細(xì)菌纖維素。木醋桿菌突變菌株C544產(chǎn)纖維素的適宜pH值范圍在5.57.0之間，在pH6.0時(shí)纖維素產(chǎn)量最高。對木醋桿菌突變株C544進(jìn)

4、行營養(yǎng)特性的研究，發(fā)現(xiàn)其能夠利用多種碳源生產(chǎn)細(xì)菌纖維素，其中以甘露醇為碳源時(shí)，纖維素產(chǎn)量最高，其次是D果糖和葡萄糖。木醋桿菌突變株C544能利用大豆蛋白陳、牛肉膏、酵母膏、蛋白陳等多種有機(jī)氮源，其中以利用大豆蛋白陳為氮源時(shí)纖維素產(chǎn)量最局。在以10%甘露醇為碳源的基礎(chǔ)培養(yǎng)基中，木醋桿菌突變株C544產(chǎn)纖維素能力達(dá)到9.33gL，是培養(yǎng)基優(yōu)化前的4.22倍。為適應(yīng)工業(yè)生產(chǎn)，選擇廉價(jià)的葡萄糖為碳源，優(yōu)化出的培養(yǎng)基配方為:5.0%(wv)葡萄糖

5、0.9%(WA)大豆蛋白陳、0.8%(wv)Na2HP04.121420及0.5%(wv)檸檬酸，pH6.0。優(yōu)化后，纖維素產(chǎn)量達(dá)到哈爾濱工業(yè)大學(xué)理學(xué)碩士學(xué)位論文AbstractBacterialCelluloseasanovelbiomaterialhasfoundamultitudeofapplicationsinpapermakingtextilemedicineandfoodindustries.Buttheapplicatio

6、nofbacterialcelluloseisrestrictedbythehighcostofproducingit.Thereforeitisnecessarytoselectandbreedhighyieldcelluloseproducingstrainsandtooptimizemediumandconditionsoffermentationsoastoincreasetheyieldofbacterialcellulose

7、andtodecreasethecost.A.xylinumC5asoriginalstrainaL9(34)orthogonaldesigningexperimentwasusedtooptimizetheconditionsofcellsuspensionpreparation.Accordingtotherangeincidenceanalysistheoptimumconditionsofcellsuspensionprepar

8、ationfortheA.xylinumC5wereconfirmed.After9daysofstaticincubation(300C)theformedcellulosepelliclewasasepticallytransferredtoasterilePetridish(12cmdiameter)towhich1.5mlofcellulasewereadded.Subsequently，cellulosewasenzymati

9、callyhydrolysed12h(30C)theinitialconcentrationofthebacteriais2.2X106m1i.ToderivemutantsfromA.xylinumC5UVmutagenesiswasused.Firstthedeathratecurvewasobtained.TimeofexposuretoUVirradiationwasdeterminedin3min.230colonieswer

10、epickedfromtheenrichmentmedia.TheamountofcellulosesynthesizedwasevaluatedvisuallyusingananalogouslygrownwildtypeA.xylinumC5asacontrol.Seventytwopotentialcelluloseoverproducingmutantswereselected.Throughscreening4strains(

11、24446898)synthesizedcelluloseinaconcentrationwhichvisiblyexceededtheoneobtainedwithparentstrainA.xylinumC5wereobtained.Contrastwiththeoriginalstrainthecelluloseyieldofthesemutantstrainswereimprovedby29.88%46.06%19.50%and

12、38.17%.Accordingtotheseresultsmutantstrain44(A.xylinumC544)waschose.OnthebaseofthesinglefactorexperimentstherangesoftemperatureforgrowthandcelluloseproductionofAcetobacterxylinumC544werewider(2531℃).Theoptimumtemperature